Method for treatment of HIV infection

ABSTRACT

A method for treatment of HIV infection includes administering at least one anti-HIV drug, such as a reverse transcriptase inhibitor, to a patient in need of such treatment and administering an extract from inflammatory tissue inoculated with vaccinia virus to the patient following the administration of the at least one anti-HIV drug. The extract maintains suppressive action on HIV replication, even if the administration of the anti-HIV drug is terminated.

BACKGROUND OF THE INVENTION

The present invention relates to a novel method for treatment of HIVinfection by using an extract from inflammatory tissue inoculated withvaccinia virus. More particularly, it relates to a combined therapy forHIV infection with anti-HIV drugs and the extract.

Acquired Immunodeficiency Syndrome (AIDS) is a disease caused byinfection of Human Immunodeficiency Virus (HIV), one species oflentivirus, which induces a progressive decrease in immune functionleading ultimately to death. At present in the United States (as ofAugust 2003), eight nucleoside/nucleotide reverse transcriptaseinhibitors, three non-nucleoside reverse transcriptase inhibitors, sevenprotease inhibitors, and one fusion inhibitor are approved as anti-HIVchemotherapeutic drugs. A therapy using a combination of these drugs,named highly active antiretroviral therapy (HAART), is commonly used andstandardized.

HAART can suppress the replication of HIV in an infected patient and canprevent the progress of HIV infection. However, it is necessary tosuppress the replication of HIV completely for several decades toprevent the development of AIDS. The discontinuation of the chemotherapyat any time following injection causes a rebound in, and repopulationof, HIV. Thus, a patient is required to receive the chemotherapycontinuously during most of his or her lifetime. It is difficult to takeanti-HIV drugs continuously because anti-HIV drugs have a largeformulation. As a result, many drugs must be taken at each dosage.Further, the anti-HIV drugs induce various and strong side effects. If apatient does not keep the dosage schedule very strictly, the treatmentmeets with failure by the induction of a drug-resistant virus.

As mentioned above, the continuous use of anti-HIV drugs for a longperiod of time in HIV infected patients, while keeping a consistentdosage schedule of almost 100%, has caused various problems, includingthe decrease of quality of life (QOL) for patients, economic burdens,and risk of long term toxicity. To avoid these problems, it is desirableto provide an alternative to continuous treatment of HIV-1 infectedpatients with conventional FDA approved anti-HIV chemotherapeutic drugregimens.

The pharmacological activities of an extract from an inflammatory tissueinoculated with vaccinia virus include: (1) analgesic, sedative,anti-stress and anti-allergic effects (Japanese Patent Laid-Open No.Sho-53-101515); (2) immuno-enhancing, anti-cancer and hepatocirrhosissuppressive effects (Japanese Patent Laid-Open No. Sho-55-87724); (3)therapeutic effect for idiopathic thrombocytopenic purpra (JapanesePatent Laid-Open No. Hei-1-265028); (4) therapeutic effects forpost-herpetic neuralgia, brain edema, dementia, and spiro-cerebellardegeneration purpra (Japanese Patent Laid-Open No. Hei-1-319422, U.S.Pat. No. 5,013,558); (5) therapeutic effects for Raynaud syndrome,diabetic neuropathy, and sequelae of myelo-optico neuropathy (JapanesePatent Laid-Open No. Hei-2-28119); (6) inhibitory effect on kallikreinproduction and improving effect of peripheral circulatory disturbance(Japanese Patent Laid-Open No. Hei-7-97336, U.S. Pat. No. 5,560,935);(7) improving effect of bone atrophy (Japanese Patent Laid-Open No.Hei-8-291077); (8) suppressive effect of nitrogen monoxide useful fortherapy of sepsis and endotoxin shock (Japanese Patent Laid-Open No.Hei-10-194978, U.S. Pat. No. 6,051,613); (9) therapeutic effect forosteoporosis (Japanese Patent Laid-Open No. Hei-11-80005); (10)therapeutic effect for AIDS by Nef action inhibiting effect orchemokine-production increasing effect (Japanese Patent Laid-Open No.Hei-11-139977 or 2000-336034); (11) therapeutic effect for ischemicdiseases such as cerebral infarction (Japanese Patent Laid-Open No.2000-16942); and (12) therapeutic effect for fibromyalgia (InternationalPCT Publication No. WO2004/039383).

An aspect of the present invention is to provide a method for treatmentof HIV infection to offer an alternative to the problems associated withthe long term use of anti-HIV drugs, such as the decrease of QOL ofpatients accompanied with therapy for HIV infection including AIDS,economical burdens, strong side effects of anti-retroviral drugs, andappearance of drug-resistant viruses. In particular, the presentinvention provides a novel adjunct method for the treatment of HIV togive persisting effectiveness to suppress HIV replication.

The present inventors have conducted various studies regarding thesuppression on HIV replication for the therapy of HIV infection. As aresult, the inventors found that after viral loads are lowered by theadministrations of an approved anti-HIV drug, the suppressive action onHIV replication can be maintained by the administration of an extractfrom inflammatory tissue inoculated with vaccinia virus, during a timeperiod during which the conventional anti-HIV drugs are terminated.

SUMMARY OF THE INVENTION

The present invention provides an alternative to continuous treatment ofHIV-1 infected patients with conventional FDA approved anti-HIVchemotherapeutic drug regimens. Thus, patients can be administered theconventional anti-HIV chemotherapeutic drugs until the level of HIV inthe blood is reduced to below detectable levels. Thereafter, thepatients can be administered pharmaceutically effective dosages of anextract prepared from tissues that have been previously injected withvaccinia virus and demonstrate readily observable inflammatoryresponses. Such skin extracts can be administered without therequirement for the conventional anti-HIV chemotherapeutic drugs andhave been shown to maintain the low to undetectable viral loads for aprolonged period of time thus providing the patients with a time periodwithout conventional anti-HIV chemotherapy and in effect giving thepatients a “drug holiday” thereby reducing the side effects andconcurrently improving QOL with an opportunity to regenerate effectiveimmunological effectiveness. In embodiments of the invention the extractmay be administered during and after treatment with at least oneanti-HIV drug. The administration of the extract may be initiated justprior to or after termination of treatment with the anti-HIV drug tomaintain substantially reduced viral load or viral level of HIV-RNA foran extended period of time even after termination of treatment with thestandard anti-HIV drug. In embodiments of the invention theadministration of the extract may be initiated when the plasma virallevel of HIV-RNA is lowered to less than a detectable limit byadministration of the standard anti-HIV drug. The extract from theinflammatory tissue prepared from rabbit skin following vaccinia virusadministration is combined with the standard anti-HIV drug such as areverse transcriptase inhibitor to reduce treatment time with thestandard anti-HIV drug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph representing the levels of plasma viral loads inrhesus macaques without anti-HIV drug therapy after SIV infection.

FIG. 2 shows a graph representing the levels of plasma viral loads inrhesus macaques with only the standard anti-HIV drug PMPA(9-R-(2-phophorylmethoxypropyl)adenine) therapy administered followingSIV infection.

FIG. 3 shows a graph representing the levels of plasma viral loads inrhesus macaques in which the administration of an extract frominflammatory tissue inoculated with vaccinia virus was initiated justbefore the termination of PMPA therapy after SIV infection.

DETAILED DESCRIPTION OF THE INVENTION

According to the protocol outlined in the present invention, thesuppressive action on HIV replication obtained by the administration ofa previously approved anti-HIV drugs may be maintained by theadministration of an extract prepared from inflammatory tissue of arabbit inoculated with vaccinia virus. Approved anti-HIV drugs may causevarious side effects and other problems and may not be administeredcontinuously for a long term period of time. Nevertheless, inembodiments of the present invention, the suppressive action on HIVreplication from the approved anti-HIV drugs may be maintained byadministration of an extract from inflammatory tissue inoculated withvaccinia virus for a certain period following the reduction stage ofvirus level, even after the administrations of anti-HIV drugs areterminated. In embodiments of the present invention, the extract may beadministered: (1) after viral loads of HIV in the blood of a patient arelowered by administration of at least one anti-HIV drug, or (2) afterthe plasma viral level of HIV-RNA is lowered to less than a detectablelimit by administration of at least one anti-HIV drug.

The extract used according to the present invention is a safe drughaving no problems such as the side effects observed in the anti-HIVdrugs which are presently used. Therefore, the method of the presentinvention for treatment of HIV infection provides an effectivealternative form of therapy to solve the problems noted above, such asthe decrease of QOL of patients accompanied with the therapy for HIVinfection including AIDS, economic burdens, strong side effects ofanti-retroviral drugs, and the appearance of a drug-resistant virus.

An extract which may be used according to the present invention is anextract containing non-proteinaceous, biofunction-regulating substancesproduced in inflammatory tissue inoculated with vaccinia virus. Thereare various reports on the bio-active substances which are produced inan inflammatory tissue inoculated with vaccinia virus, methods forextracting such substances from diseased tissues, and thepharmacological activities thereof. The extracts, manufacturing methodsof the extracts, and preferred doses thereof are disclosed in the patentpublications discussed above. In addition, U.S. Pat. Nos. 5,013,558,5,560,935, 6,051,613, and 6,165,515 are incorporated herein by referencein their entireties as to the extracts and active ingredients,manufacturing methods of the extracts, and doses disclosed therein.

In embodiments of the present invention, animals for preparing theinflammatory tissues by inoculation of vaccinia virus include but arenot limited to rabbits, cows, horses, sheep, goats, monkeys, rats, mice,guinea pigs, hamsters, swine, chickens, and the like.

The animal tissues used in the present invention may be culturedtissues, cultured cells or inflammatory tissues of human or animalorigin which are infected with vaccinia virus, or chorio-allantoicmembranes of embryonated eggs infected with virus. Examples of suchcultured cells which may be utilized are various tissues (e.g., humanhemocytes and placentae) and the cultured cells of various tissues suchas kidney, skin, testis, lung, muscle, adrenal gland, thyroid gland,brain, nerve cells and hemocytes of the above-mentioned animals andembryos thereof. In preferred embodiments, the inflammatory tissues areinflammatory rabbit skin.

A commercially available drug preparation of an extract frominflammatory rabbit skin inoculated with vaccinia virus which may beemployed in the present invention is described at pages 2499-2501 of“Drugs in Japan, Ethical Drugs” (27th ed., (2004)), edited by JapanPharmaceutical Information Center, published by Yakugyo Jiho Co., Ltd.As described therein, this preparation is a drug containingnon-proteinaceous active substances extracted and isolated frominflammatory skin of rabbits inoculated with vaccinia virus. This drughas been used for low back pain, neck-shoulder-arm syndromes,periarthritis scapulohumeralis, osteoarthritis, symptomatic neuralgia,itching accompanied with skin disorders (such as eczema, dermatitis andurticaria), allergic rhinitis, sequelae of subacutemyelo-optico-neuropathy (such as coldness, pain andparesthesia/dysesthesia), post-herpetic neuralgia, and the like. Thedrug is approved as an ethical drug in the forms of injections(subcutaneous, intramuscular and intravenous) and in the form of tabletsthat are commercially available. This drug preparation is available inJapan and has the tradename NEUROTROPIN.

An extract from inflammatory tissue inoculated with vaccinia virus foruse in the present invention may be produced by inoculating an animalwith vaccinia virus to cause inflammation. The inflammatory tissues arefinely cut; an extracting medium is added thereto; and tissue residuesare removed. A procedure to remove proteins is carried out in whichactive ingredients are adsorbed to the adsorbent, and the adsorbedingredients are eluted from the adsorbent. For example, an extract frominflammatory tissue inoculated with vaccinia virus may be produced bythe following steps:

(a) An animal is inoculated with vaccinia virus and inflammatory tissuessuch as the skin is removed and finely cut. An extracting medium such aswater, phenol water, saline, or phenol-added glycerin water is addedthereto and then filtration or centrifugation is conducted to give anextracted solution (filtrate or supernatant).

(b) The extracted solution is adjusted to acidic pH and heated to removeproteins. The protein-removed solution is adjusted to alkaline pH andheated again, and then filtered or centrifuged.

(c) The resulting filtrate or supernatant is adjusted to acidic pH andadsorbed to an adsorbent such as active carbon or kaolin.

(d) An extracting solvent such as water or the like is added to theadsorbent and adjusted to alkaline pH to elute adsorbed ingredients togive an extract from inflammatory tissue inoculated with vaccinia virus.Then, if desired, the extraction may suitably be evaporated to drynessunder reduced pressure or freeze-dried to make dried materials.

In embodiments, the inflammatory tissues may be removed, finely cut, andmade into an emulsified suspension by adding 1 to 5 times as muchextracting solvent thereto. Examples of the extracting solventapplicable include distilled water, physiologically saline solution,weakly acidic to weakly basic buffers, and the like. If necessary,stabilizers such as glycerol; antibacterial/antiseptic agents such asphenol; and inorganic salts such as sodium chloride, potassium chloride,or magnesium chloride may be added thereto. At that time, the extractioncan be facilitated with a treatment by freezing/melting, ultrasonicwaves, cell membrane dissolving enzymes or surface-active agents tocause cell destructions.

The resulting milky extract may be filtered or centrifuged to removetissue residues and then proteins may be removed therefrom. Removal ofproteins can be carried out by known methods, for example, heating;treatments with protein denaturing agents such as acids, bases, urea,guanidine, organic solvents such as acetone, surface-active agents, andthe like; isoelectric precipitation; salting-out; and the like. Then,the precipitated proteins may be removed, for example, by filtrationusing filter paper (cellulose, nitrocellulose, and the like), glassfilter, Celite or a Seitz filter, ultrafiltration, centrifugation, andthe like.

The obtained extract containing ingredients may be adjusted to an acidicpH, preferably to a pH of 3.5 to 5.5, by acids such as hydrochloricacid, sulfuric acid, and hydrobromic acid, and may be adsorbed with anadsorbent. As an adsorbent, activated charcoal, kaolin, and the like maybe employed. The adsorbents may be added to the extract followed bystirring or the extract may be passed through a column filled with theadsorbents, whereby active ingredients can be adsorbed.

To elute the ingredients from the adsorbents, an eluting solvent may beadded to the adsorbents and eluted at room temperature or with heatingto some extent or with stirring. The adsorbents may be removed byconventional means such as filtration and centrifugation to complete theelution. As an eluting solvent, water, methanol, ethanol, isopropanol ora mixture thereof which may be adjusted to basic pH may be employed.Preferably, water adjusted to a pH of 9 to 12 can be used.

The extract (eluted solution) produced as above can be prepared todesired formulations for raw materials or medicines. For example, thesolution may be adjusted to neutral pH to prepare raw materials ofdrugs, and may be adjusted to desired concentrations by condensation ordilution. Furthermore, in order to prepare an injection, the solutionmay be prepared to an isotonic solution the same as saline. The solutionmay be prepared to solid preparations available for raw materials oftablets and the like by concentration to dryness or lyophilization.

As a method of administration, oral and other administrations such assubcutaneous, intramuscular and intravenous administrations may be used.The dosage to be utilized is dependent on the kind of extractionprocedure utilized from the inflammatory tissue inoculated with vacciniavirus. The dose which is approved in the commercially availablepreparation according to “Drugs in Japan, Ethical Drugs” (page 2499) is,principally, 16 NU per day and 3.6-7.2 NU per day by oral administrationand by injection, respectively. However, the dose or pharmaceuticallyeffective amount may be appropriately increased or decreased dependingupon the type of the disease, degree of seriousness, individualdifference in the patients, method of administration, period ofadministration, and the like (NU: Neurotropin unit). Neurotropin unit isdefined by ED₅₀ value of analgesic effect measured by a modifiedRandall-Selitto method using SART-stressed mice. The SART-stressed miceare chronic stressed animals showing a lowered pain threshold than anormal animal. One NU indicates the activity of 1 mg of analgesicingredients in Neurotropin preparations when the ED₅₀ value is 100 mg/kgof the preparation.

As an anti-HIV drug used in the method for treatment of the presentinvention, any drugs having a reducing action on viral loads of HIV inblood can be used. In embodiments, nucleoside/nucleotide analoguereverse transcriptase inhibitors such as Abacavir (ABC), Didanosine(ddI), Emtricitabine (FTC), Lamivudine (3TC), Stavudine (d4T), Tenofovir(TDF), Zalcitabine (ddC) and Zidovudine (AZT); non-nucleoside reversetranscriptase inhibitors such as Delavirdine (DLV), Efavirenz (RFV) andNevirapine (NVP); protease inhibitors such as Amprenavir (APV),Atazanavir (ATV), Indinavir (IDV), Ritonavir (RTV), Lopinavir/Ritonavir(LPV/RTV), Nelfmavir (NFV) and Saquinavir (SQV); and fusion inhibitorssuch as Enfuvirtide (T20) can be employed as conventionalanti-retroviral drugs which are already approved for use in HIVinfection in the United States. In preferred embodiments, a combinationof the anti-HIV drugs may be used, however, the method of the presentinvention is not limited thereto. The dose or pharmaceutically effectiveamount, administration route, the number of administrations, and thelike of the HIV-drugs can be determined according to various conditions.

The anti-HIV drugs and the extract from inflammatory tissue inoculatedwith vaccinia virus may each be used in pharmaceutically effectiveamounts for treating humans or animals, such as mammals, in need oftreatment for HIV infection.

The following non-limiting examples illustrate manufacturing methods forproducing an extract from inflammatory tissue inoculated with vacciniavirus, and pharmacological studies. All parts, percentages and ratiosare by weight, all temperatures are in ° C., and all reactions areconducted at about atmospheric pressure and room temperature unlessindicated to the contrary. In the following Examples 2 and 3, thedryness in vacuo is conducted in the final steps. However, thisprocedure is for making tablets and, therefore, is not indispensable.The results of pharmacological studies show a persisting effect forsuppressing the proliferation of retrovirus for treatment of HIVinfection:

EXAMPLE 1

Skins of healthy adult rabbits were inoculated with vaccinia virus tocause inflammation. The inflammatory skins were removed, finely cut andphenol water was added thereto. The mixture was filtered with pressure,and the resulting filtrate was adjusted to pH 5 with hydrochloric acidand then heated at 90-100° C. for 30 minutes. Proteins were removed byfiltration, the filtrate was adjusted to pH 9 with sodium hydroxide,further heated at 90-100° C. for 15 minutes and filtered. The filtratewas adjusted to about pH 4, stirred for 2 hours after adding 2% ofactivated charcoal, and centrifuged. The resulting activated charcoalwas mixed with water, adjusted to pH 10 with sodium hydroxide, stirredat 60° C. for 1.5 hours and centrifuged to give a supernatant. Theactivated charcoals precipitated by centrifugation were mixed withwater, adjusted to pH 11 with sodium hydroxide, stirred at 60° C. for1.5 hours and centrifuged to give a supernatant. Both of thesupernatants obtained were combined and neutralized with hydrochloricacid to give an extract from inflammatory tissue inoculated withvaccinia virus. In the following pharmacological studies, the extractwas adjusted to appropriate concentrations to be used.

EXAMPLE 2

Skins of healthy adult rabbits were inoculated with vaccinia virus tocause inflammation. The inflammatory skins were aseptically removed,finely cut and phenol-added glycerin water was added thereto. Themixture was ground using a homogenizer to prepare an emulsion. Theemulsion was filtered with centrifugation, and the resulting filtratewas adjusted to pH 4.8-5.5 with hydrochloric acid, heated at 100° C.with a steam flow and then filtered. The filtrate was further filteredwith Seitz filter, adjusted to pH 9.2 with sodium hydroxide, heated at100° C. and filtered. The filtrate was adjusted to pH 4.5, stirred for1-5 hours after adding 1.5% of activated charcoal, and filtered. Theactivated charcoal was mixed with water, adjusted to pH 9.4-10 withsodium hydroxide, stirred for 3-5 hours and filtered. The resultingfiltrate was neutralized with hydrochloric acid and dried in vacuo.

EXAMPLE 3

Skins of healthy adult rabbits were inoculated with vaccinia virus toactivate or stress the tissues. The activated skins were asepticallyremoved, finely cut and water was added thereto. The mixture was groundusing a homogenizer to prepare an emulsion. The emulsion was filteredwith pressure, and the resulting filtrate was adjusted to pH 5.0 withhydrochloric acid and heated at 100° C. with a steam flow. Proteins wereremoved by filtration, the filtrate was adjusted to pH 9.1 with sodiumhydroxide, heated at 100° C. and filtered. The filtrate was adjusted topH 4.1, stirred after adding 2% of activated charcoal, and the mixturewas filtered to obtain a filtrate and a first batch of recoveredactivated charcoal. To the filtrate was added 5.5% of activated charcoaland the mixture was stirred for 2 hours, and filtered to obtain a secondbatch of recovered activated charcoal. The first batch of recoveredactivated charcoal was mixed with water, adjusted to pH 9.9 with sodiumhydroxide, stirred at 60° C. for 1.5 hours and filtered. Water was thenadded to the first batch of the activated charcoal and to the secondbatch of activated charcoal. The pH of each batch was then adjusted topH 10.9 with sodium hydroxide, and each batch was stirred at 60° C. for1.5 hours and then filtered. The resulting filtrates were combined,neutralized with hydrochloric acid, desalted using electrodialysis withmembrane (molecular weight: 100), and dried in vacuo.

EXAMPLE 4 Pharmacological Study

A correlative pharmacological study regarding a method for treatment ofHIV infection according to the present invention, namely ananti-retroviral action, was conducted. The pharmacological study wasperformed to determine the effectiveness of an extract from inflammatorytissue inoculated with vaccinia virus to influence the levels of viralrebound in SIV (Simian Immunodeficiency Virus)-infected monkeysfollowing a standard 28 day single cycle anti-retroviral drug therapy.

This pharmacological study included a total of 3 groups of rhesusmacaques (Macaca mulatta) of Indian origin with 4 monkeys included ineach group. Group 1 included the monkeys (Virus control) that were nottreated by an anti-retroviral drug after SIVinfection. The monkeys inGroup 2 (PMPA control) received a single 28 day cycle of dailyadministration of an anti-retroviral drug. The monkeys in Group 3(Extract treatment) were administered an extract prepared frominflammatory tissue inoculated with vaccinia virus following a single 28day cycle of daily administratoin of the same anti-retroviral drug asGroup 2 monkeys. For each group, the studies were performed as follows:

(1) Virus Infection (All Groups)

A single large batch of SIVmac239 was grown in day 3 rhesus PHA blasts.The supernatant fluids were ultracentrifuged, and the resulting viruswas purified on a sucrose gradient and then pelleted. The pelleted viruswas then resuspended in 1.0 mL of PBS (phosphate buffered saline) andtermed virus stock. The level of virus was determined and the level ofreplication competent virus was titrated.

The stock virus was then diluted so as to contain approximately 200AID₅₀ (50% animal infectious dose) in a volume of 1.0 mL, and eachanimal in each group was injected intravenously with 1.0 mL of the virussolution to infect with SIV.

(2) Determination of Viral Load Set Point (All Groups)

Following SIV infection, each animal was bled approximately 1.0 mL ondays 0, 7, 14, 21, 28, 42 and 56 post infection and the viral level wasquantified by real time PCR. The viral copy number per ml was recordedand viral load set point was determined as a value that reaches aplateau following increased viral level following initial viral loadspike.

(3) PMPA Therapy (Groups 2 and 3)

PMPA (9-R-(2-phophorylmethoxypropyl)adenine) is the standard drug foranti-retroviral chemotherapy of SIV infected non-human primates. Themonkeys of Group 2 (PMPA control) and Group 3 (Extract treatment) weresubcutaneously administered with PMPA at a dose of 30 mg/kg daily for 28days soon after they reached viral load set point and the levels ofplasma viral loads was determined. PMPA administration at a dose of 30mg/kg daily for 28 days was decided because it was previously determinedto be an effective dose regimen that leads to a reduction of plasma andcellular viral loads to almost undetectable levels.

(4) Administration of an Extract from Inflammatory Tissue Inoculatedwith Vaccinia Virus (Group 3)

An extract from inflammatory tissue inoculated with vaccinia virusproduced in Example 1 was administered to the monkeys of Group 3(Extract treatment) to determine the effectiveness of the extract toinfluence the level of plasma viremia following the anti-retroviral drugtherapy. The extract from inflammatory tissue inoculated with vacciniavirus was adjusted to the appropriate concentration and sonicated for 20minutes at 60° C. and filtered through a 0.45-μm filter. It was thenadministered subcutaneously at a dose of 3.3 NU/kg daily for 60 daysinitiated at 2 days prior to the termination of PMPA therapy and thelevel of plasma viral loads were continuously monitored.

(5) Results

The graphs representing the levels of plasma viral loads after theinfection of SIV of each group according to the above-mentionedprocedures are shown for each monkey in FIGS. 1-3.

The plasma viral loads after infection with SIV ranged from 100,000 to10 million viral copies per mL at peak and then reached a set point 6-8weeks post infection. The viral loads in Group 1 (Virus control) withoutany therapy at this point stayed at the set point (FIG. 1). However,PMPA therapy led to a marked reduction in plasma viral loads to between100 to 1000 viral copies per mL by day 28 in Group 2 (PMPA control) andin Group 3 (Extract treatment).

Thereafter, in Group 2 (PMPA control) in which no further treatment wascontinued following the termination of PMPA therapy, it appeared thatviral loads rebounded soon after the discontinuation of PMPA therapy(FIG. 2). On the other hand, in Group 3 (Extract treatment) in which theadministration of an extract from inflammatory tissue inoculated withvaccinia virus was initiated just before the termination of PMPAtherapy, it was possible to keep the suppression of viral loads for along period of time even if the administration was stopped after only 60days (FIG. 3).

As apparent from the results of the above pharmacological studies, it isdemonstrated that, according to the method of the present invention fortreatments of HIV infection, the suppressive action on HIV replicationcan be maintained by the administration of an extract from inflammatorytissue inoculated with vaccinia virus for a certain period following theachievement of reduced viral loads induced by the administrations ofanti-HIV drugs (i.e., the anti-HIV drugs were only administered dailyfor 28 days). The longer the approved anti-HIV drugs are used, the moreproblems have been noted to occur, such as strong side effects,appearance of resistant virus to the drug, decrease of QOL of patients,and economical burdens.

Therefore, since the suppressive effect on HIV proliferation caused byanti-HIV drugs can be maintained by use of an extract from inflammatorytissue inoculated with vaccinia virus for a short term, which has noproblems such as side effects observed in the usual anti-HIV drugs, themethod of the present invention for treatment of HIV infection is auseful adjunct therapy to solve the problems as mentioned above.

1. A method for treatment of HIV infection comprising: administering atleast one anti-HIV drug to a patient in need of such treatment; andadministering an extract from inflammatory tissue inoculated withvaccinia virus to the patient following the administration of the atleast one anti-HIV drug.
 2. A method according to claim 1 wherein the atleast one anti-HIV drug is at least one drug selected from the groupconsisting of nucleoside/nucleotide reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, protease inhibitors,and fusion inhibitors.
 3. A method according to claim 1 wherein the atleast one anti-HIV drug comprises a reverse transcriptase inhibitor. 4.A method according to claim 3 wherein the reverse transcriptaseinhibitor is at least one member selected from the group consisting ofAbacavir (ABC), Didanosine (ddI), Emtricitabine (FTC), Lamivudine (3TC),Stavudine (d4T), Tenofovir (TDF), Zalcitabine (ddC), Zidovudine (AZT),Delavirdine (DLV), Efavirenz (RFV), and Nevirapine (NVP).
 5. A methodaccording to claim 1 wherein the inflammatory tissue comprises skintissue of a rabbit.
 6. A method according to claim 1 wherein viral loadsof HIV in the blood of the patient are lowered by administration of theat least one anti-HIV drug.
 7. A method according to claim 1 wherein theplasma viral level of HIV-RNA is lowered to less than a detectable levelby the administration of at least one anti-HIV drug.
 8. A methodaccording to claim 1 wherein the administration of at least one anti-HIVdrug is terminated and the extract maintains suppressive action on HIVreplication.
 9. A method for treatment of HIV infection comprising theadministration of an extract from inflammatory tissue inoculated withvaccinia virus to a patient in need of such treatment after viral loadsof HIV in blood are lowered by the administration of at least oneanti-HIV drug, or after the plasma viral level of HIV-RNA is lowered toless than a detectable limit by the administration of at least oneanti-HIV drug.
 10. A method according to claim 9 wherein theadministration of an extract from inflammatory tissue inoculated withvaccinia virus to a patient in need of such treatment is after theplasma viral level of HIV-RNA is lowered to less than a detectable limitby the administration of at least one anti-HIV drug.
 11. A methodaccording to claim 9 wherein the inflammatory tissue is skin tissue ofrabbit.
 12. A method according to claim 9 wherein the at least oneanti-HIV drug comprises a reverse transcriptase inhibitor.
 13. A methodaccording to claim 10 wherein the inflammatory tissue is skin tissue ofrabbit.
 14. A method according to claim 10 wherein the at least oneanti-HIV drug comprises a reverse transcriptase inhibitor.
 15. A methodaccording to claim 9 wherein said at least one anti-HIV drug comprisesat least one member selected from the group consisting of Abacavir(ABC), Didanosine (ddI), Emtricitabine (FTC), Lamivudine (3TC),Stavudine (d4T), Tenofovir (TDF), Zalcitabine (ddC), Zidovudine (AZT),Delavirdine (DLV), Efavirenz (RFV), Nevirapine (NVP), Amprenavir (APV),Atazanavir (ATV), Indinavir (IDV), Ritonavir (RTV), Lopinavir/Ritonavir(LPV/RTV), Nelfinavir (NFV), Saquinavir (SQV), and Enfuvirtide (T20) 16.A method for treatment of HIV infection comprising reducing the viralload of HIV in the blood or reducing the plasma viral level of HIV-RNAin a patient by the administration of a pharmaceutically effectiveamount of at least one anti-HIV drug, and administering apharmaceutically effective amount of an extract from inflammatory tissueinoculated with vaccinia virus while said viral load of HIV or virallevel of HIV-RNA is reduced.
 17. A method according to claim 16 whereinsaid extract is administered during and after treatment with said atleast one anti-HIV drug.
 18. A method according to claim 16 wherein saidadministration of said extract is initiated just prior to termination oftreatment with said at least one anti-HIV drug.
 19. A method accordingto claim 16 wherein said administration of said extract is initiatedafter termination of treatment with said at least one anti-HIV drug. 20.A method according to claim 16, wherein said administration of saidextract is initiated when the plasma viral level of HIV-RNA is loweredto less than a detectable limit by administration of the at least oneanti-HIV drug, the inflammatory tissue is skin tissue of rabbit, and theanti-HIV drug comprises a reverse transcriptase inhibitor.